Adiponectin is a secretory protein composed of 244 amino acids, which was identified in 1996 by Matsuzawa (Department of Internal Medicine and Molecular Science, Osaka University; Sumitomo Hospital at present) et al. as a gene product of a gene apM1 (adipose most abundant gene transcript) specifically expressed in adipose tissues (non-patent references 1 and 2). Adiponectin is contained at a high concentration (about 1 μg/mL to several tens of μg/mL) in normal human blood. Although adiponectin is specifically secreted from adipocytes, obese persons show a significantly low concentration thereof in blood, and adiponectin is lowered in patients suffering from coronary diseases or type II diabetes, particularly diabetic macroangiopathy. Adiponectin may be regarded as a molecule involved in insulin resistance and arteriosclerosis. It is important for preventing coronary diseases to measure adiponectin rapidly and accurately.
As a method for measuring adiponectin, an immunological measuring method using an antibody specific to a substance to be analyzed is known (non-patent reference 3). In the immunological measuring method, a radioimmunoassay or an enzyme immunoassay, in which a radioactive substance or an enzyme is used as a label, is utilized to measure an immunocomplex formed by an antigen-antibody reaction (non-patent references 4-7 and patent references 1 and 2). In the radioimmunoassay, facilities for measurement are limited, since a radioactive substance is used. Further, it is generally necessary to dilute a sample to 1/500, and it takes 20 to 24 hours to carry out the measurement. In the enzyme immunoassay, it is generally necessary to pretreat a sample with sodium dodecyl sulfate (SDS) and to predilute a sample to approximately 1/5000, and it takes 2 hours or more to carry out the measurement. As above, the conventional adiponectin measurements need special facilities, complicated procedures, and a long measuring time.
When blood, which reflects a pathosis faithfully, is used as a sample in the above conventional methods, complicated procedures and a long measuring time are needed, and thus, the methods are not suitable for a general purpose assay or a multisample assay. It is desired to develop a measuring reagent for an automatic analysis in which the analysis can be performed rapidly and conveniently, and facilities therefor are not limited.
More particularly, the patent reference 1 discloses an ELISA method for analyzing adiponectin, in which a polyclonal antibody and a monoclonal antibody prepared by using as an immunogen adiponectin expressed in an Escherichia coil by genetic recombination techniques are used. The patent reference 1 disclosed that when the ELISA method was used to measure a concentration of adiponectin contained in normal human plasma, without a pretreatment of the plasma sample, the measured value was lower than that previously predicted from a result obtained by Western blotting. As the reason for this, the patent reference 1 discloses a possibility that a site to be recognized by the antibody may be masked, since adiponectin in blood is assembled with other plasma components to form a macromolecule of 290 kDa or more. In the ELISA method disclosed in the patent reference 1, adiponectin in plasma can be measured by diluting the plasma to 1/10 with an SDS-containing buffer, boiling the diluted plasma for 5 minutes, diluting the boiled plasma to approximately 1/5000 as the final concentration, and measuring the 1/5000-diluted plasma. That is, the ELISA method disclosed in the patent reference 1 needs the pretreatment (the heat treatment in the presence of SDS) and the predilution of a sample.
As an ELISA method for analyzing adiponectin which does not need such a pretreatment, the patent reference 2 discloses an ELISA method in which one or more monoclonal antibodies which specifically react with a naturally-occurring adiponectin in blood (particularly, a monoclonal antibody which specifically reacts with a trimeric structure of adiponectin and/or a naturally-occurring adiponectin having a structure in which the trimers are further assembled) is used. According to the disclosure in the patent reference 2, it is known that adiponectin in blood forms a structure in which 4 or 6 trimers composed of 3 monomers are assembled (non-patent reference 8), and the pretreatment of a sample is not necessary in the ELISA method disclosed in the patent reference 2, since one or more monoclonal antibodies specific to a naturally-occurring adiponectin are used. However, the predilution of a sample is an essential step in the ELISA method disclosed in the patent reference 2. As an assay, the patent reference 2 exemplifies, for example, a solid phase method, a competitive method, an agglutination method, a turbidimetric method, and a sandwich enzyme immunoassay, and discloses that ELISA is most preferable. Examples described in the patent reference 2 do not include embodiments other than ELISA.
(non-patent reference 1) Biochemical and Biophysical Research Communications, (U.S.A.), 1996, vol. 221, p. 286-289
(non-patent reference 2) Gene, (Netherlands), 1997, vol. 190, p. 227-235
(non-patent reference 3) Hiroshi Hirose et al., No. 163, “Kessei adiponectin noudo to insulin teikousei: kenjyojin oyobi 2-gata tonyoubyou kanjya ni okeru kentou”, “Meeting of the 75th Japanese endocrinology association study, Abstracts”, The Japan Endocrine Society, 2002, p. 118(non-patent reference 4) Yasuichi Ohmoto et al., “Adiponectin no ELISA kit ni tuite”, Bio Clinica, 2002, vol. 17, p. 156-159(non-patent reference 5) Yasuichi Ohmoto et al., “Adiponectin ELISA kit no kaihatsu to kecchu sonzai youshiki no kaiseki”, Medical Science Digest, 2002, vol. 28, No. 12, p. 40-43(non-patent reference 6) Arteriosclerosis, thrombosis, and vascular biology, (U.S.A.), 2003, vol. 23, p. 85-89(non-patent reference 7) Circulation, (U.S.A.), 2003, vol. 107, p. 671-674(non-patent reference 8) Journal of Biochemistry, 1996, vol. 120, p. 803-812(patent reference 1) WO 99/21577(patent reference 2) WO 03/016906